Use of tumor dissociation reagent in flow cytometry

ABSTRACT

The present disclosure relates to a dissociation reagent for tumor tissues. The dissociation reagent does not contain collagenase or trypsin but further contains hyaluronidase or a mixture of hyaluronidase and DNase I. The present disclosure also relates to use of the dissociation reagent in dispersing tumor tissues and detecting expression level of molecular markers on cell surface by flow cytometry. The dissociation reagent of the present disclosure does not cause degradation of molecular markers on cell surface such as CD8, PD-1, Tim-3, Lag-3 and the like, thus does not affect downstream assays.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to Chinese Application No.201610235031.4 filed on Apr. 15, 2016, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to a tumor dissociation reagent useful inflow cytometry for dispersing a clinical solid tumor tissue into asingle cell suspension and for protecting the surface marker from beingdegraded.

BACKGROUND

In the analysis of the cell biological characteristics of tumor tissue(such as the detection of cell epitope, etc.), preparation in tissueblocks of the sample into a single cell suspension is required, so as toobtain high-yield cells, and integrity of cells and epitopes is intactand can be used directly in downstream experiments. Only when variouscell components of the sample tissue are in a single cell state, avariety of detection and analysis of cell effects can be effectivelycarried out, while the quality of cell suspension is closely related todigestive fluid formulations and digestion methods. The preparationmethods of single cell suspension commonly used are chemical, enzymaticand physical methods.

The cell-to-cell linkages such as collagenase, elastic fiber,mucopolysaccharide, tight junction proteins and the like are mainlydestructed for cell dispersion in enzymatic method. Trypsin,collagenase, hyaluronidase and the like are the commonly used enzymes.In prior art, it is stated that a variety of tissues including tumortissue, skeletal muscle, spleen, lung, nerve tissue, epidermis, laminapropria, mouse heart, neonatal neurons, embryoid bodies and the like maybe treated with commercial dissociation kits such as the Miltenyi TissueDissociation Kit; and according to the difference in the sensitivity ofthe cell surface antigen to enzymatic digestion, different enzymereaction systems are designed for preventing the cell surface antigendestruction, so as not to affect downstream experiments. In fact, thereare some molecular markers on cell surface that are degraded orpartially degraded in practices, making it impossible to detectaccurately.

In fact, some commonly used components of the enzymatic reagent willaffect the detection of cell surface molecular markers. For example,Trypsin has a strong ability to disperse cells with short action time.The use of trypsin in preparation of single cell suspension results inhigh yield, but the conditions of action required by trypsin arecomplex. In addition, Trypsin may damage the cell surface antigen andeven cells. For example, trypsin-treated mouse thymus cell surfacereceptors CD4 and CD8 are digested by trypsin digestion (ThomasBarthlott, Rebecca J. Wright and Brigitta Stockinger. J Immunol, 1998.161:3992-3999). Therefore, trypsin is suitable for detection ofintracellular antigens but not for cell surface antigens, especiallyweakly expressed antigens.

Immunological checkpoint proteins are key targets in tumor therapy andplay a very important role in immunotherapy, thus may be a powerfulweapon for conquering cancer. Therefore the accuracy of detection isrequired for diagnosis and treatment of cancer.

In the process of diagnosis and determination of the following treatmentregimen (especially targeted therapy) for clinical cancer patients, flowcytometry has an incomparable advantage in determination of subdivisionand cell surface markers, because it can simultaneously detect multiplemarkers on a single cell. However, the tumor tissue needs to be treatedwith a digestive enzyme into a single cell suspension before it can beused for subsequent flow cytometry. In the present disclosure, we havefound that the current commercial human tumor digestive agents ordigestive agents commonly used such as collagenases all have asignificant impact on the expression level of various proteins concernedin the present disclosure (including immunological checkpoint proteinssuch as Tim-3 and Lag3), which undoubtedly increased the risk ofmisdiagnosis of the disease and easily lead to wrong treatment.Therefore, the digestive effect of dissociation reagent on tumor tissuesand the protective effect thereof on cell surface antigen remain to beimproved. It is important to find a digestive enzyme or mixture that iseffective in digesting human tumor tissues and does not affect theexpression of surface markers.

SUMMARY

In one aspect, the present disclosure relates to a tumor dissociationreagent.

Wherein, the tumor dissociation reagent which does not comprisecollagenase but comprises hyaluronidase, said tumor dissociation reagentdoes not degrade or partially degrade membrane surface receptor.

The present disclosure also relates to a tumor dissociation reagentwhich further does not comprise trypsin and collagenase and compriseshyaluronidase, wherein the tumor dissociation reagent does not degradeor partially degrade membrane surface receptor.

Wherein, the concentration range of the hyaluronidase described in anembodiment of the present disclosure is preferably 1 mg/mL or less, andthe concentration of hyaluronidase is more preferably 1 μg/mL to 1mg/mL.

The present disclosure further relates to the preceding tumordissociation reagent, further comprising DNase I.

Wherein, the concentration range of the DNase I described in the presentdisclosure is preferably 50 μg/mL or less, and the concentration ofDNase I is more preferably 1 μg/mL to 50 μg/mL.

In one embodiment, the tumor dissociation reagent does not degrade orpartially degrade membrane surface receptor, wherein said membranesurface receptor is at least one checkpoint receptor selected from thegroup consisting of receptor CD8, PD-1, PD-L1, TIM-3 and LAG-3 protein.

In one embodiment, the proceeding membrane surface receptor is acheckpoint receptor.

In a further aspect, the present disclosure provides use of said tumordissociation reagent in detecting protein expression level of animmunological checkpoint marker in tumor tissue.

In one embodiment of the present disclosure, the preceding tumordissociation reagent further comprises DNase I.

In one embodiment of the present disclosure, the tumor tissue includestumor infiltrating immune cell.

In an embodiment of the present disclosure, the protein expression intumor tissue is detected by flow cytometry.

In an embodiment of the present disclosure, the protein is membranesurface receptor for checkpoint.

In an embodiment of the present disclosure, in flow cytometry of tumortissue, said membrane surface receptor is at least one checkpointreceptor selected from the group consisting of CD8, PD-1, PD-L1, TIM-3and LAG-3 protein.

In a further aspect, the present disclosure relates a kit for tumordissociation comprising the tumor dissociation reagent of the presentdisclosure.

In an embodiment of the present disclosure, the preceding tumordissociation reagent further comprises DNase I.

The present disclosure also relates to use of the kit in detectingprotein expression in tumor tissue by flow cytometry.

In a further aspect, the present disclosure also provides a preparationmethod, wherein the method may prevent degradation of immunologicalcheckpoint markers in tumor tissue, wherein cells are treated with thetumor dissociation agent disclosed by the present disclosure.

In one embodiment of the present disclosure, a method of tumordissociation for flow cytometry comprises dissolving a tumor tissue withthe tumor dissociation reagent of the present disclosure.

In one embodiment of the present disclosure, the tumor tissue furtherincludes tumor infiltrating immune cell.

Advantageous Effects of the Invention

The benefit of the present disclosure resides in the establishment of atumor dissociation reagent which does not comprise collagenase ortrypsin and does not degrade or partially degrade membrane surfacemarker in tumor tissue, therefore the tumor dissociation reagent isuseful in flow cytometry for detecting the expression level of proteinsin tumor tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart showing the checkpoint proteins PD-1, Tim-3, andLag-3 in helper T cells (CD4+) treated with different digestive enzymes.

FIG. 2 is a flow chart showing the checkpoint proteins PD-1, Tim-3, andLag-3 in cytoxic T cells (CD8+) treated with different digestiveenzymes.

FIG. 3 is a graph showing the percentages of cell surface checkpointproteins after different digestive enzymes treatment.

FIG. 4 is a graph showing the percentages of cell surface checkpointproteins PD-1, Tim-3 and Lag-3 after treatment with two-enzyme mixture.

FIG. 5 is a flow chart showing the cell surface checkpoint proteins CD4and CD8 after treatment with different digestive enzymes.

FIG. 6 is a graph showing the cell yield after digestion with differentenzymes.

DETAILED DESCRIPTION

The present disclosure is further described by the specific embodimentsand experimental results. Although specific terms are used hereinafterfor the purpose of clarity, these terms are not to be limiting the scopeof the present disclosure.

As used herein, the term “dissociation reagent” refers to an enzymaticdigestion reagent, and in the present disclosure, a tumor dissociationreagent refers to an enzymatic digestion reagent that digests tumortissue into a single cell suspension with an enzyme digestion solution.

As used herein, the term “membrane surface receptor” refers to onemolecule or a class of molecules on cell surface that may recognize,bind to a specific biologically active substance (referred to as aligand), and the resulting complex may activate and initiate a series ofphysical and chemical changes that lead to the final biological effectsof the substance. Changes in the various factors of the cell environmentresult in corresponding changes of the physiological processes withinthe cell through the role of the cell membrane receptors.

The experimental methods in the following examples, unless otherwisespecified, are conventional methods.

EXAMPLES Example 1 Preparation of Digestive Enzyme Reagents

Dissociation buffer system of Hyaluronidase: 50 μL of a solution ofhyaluronidase with an initial concentration of 10 mg/mL (Hyaluronidase,available from Sigma, Cat. No. H3506) was added to 4.95 mL of DMEMmedium (the final concentration of hyaluronidase is 100 μg/mL) and thenformulated into 5 mL of dissociation reagent for tumor tissues.

Dissociation buffer system of Collagenase D: 500 μL of a solution ofcollagenase D (Collagenase D, available from Roche Corporation, Cat. No.11088882001) with an initial concentration of 10 mg/mL was added to 4.5mL of DMEM medium (the final concentration of collagenase D is 1 mg/mL)and then formulated into 5 mL of dissociation reagent for tumor tissues.

Dissociation buffer system of DNase I: 50 μL of a solution of DNase I(DNase I, available from Sigma Corporation, Cat. No. DN25-1G) with aninitial concentration of 5 mg/mL was added to 4.95 mL of DMEM medium(the final concentration of DNase I is 0.05 mg/mL) and then formulatedinto 5 mL of dissociation reagent for tumor tissues.

Dissociation buffer system of three-enzyme mixture: 50 μL of a solutionof hyaluronidase at the initial concentration of 10 mg/mL, 500 μL of asolution of collagenase D with an initial concentration of 10 mg/mL and50 μL of a solution of DNase I with an initial concentration of 5 mg/mLwere added to 4.4 mL of DMEM medium and then formulated into 5 mL ofdissociation reagent for tumor tissues.

Dissociation buffer system of two-enzyme mixture: 50 μL of a solution ofhyaluronidase with an initial concentration of 10 mg/mL and 50 μL of asolution of DNase I with an initial concentration of 5 mg/mL were addedto 4.9 mL of DMEM medium and then formulated into 5 mL of dissociationreagent for tumor tissues.

Dissociation buffer system of Miltenyi Human Tumor Dissociation Kit(Human tumor dissociation kit, Cat. No. 130-095-929): according to theinstructions, the storage solution of digestive enzymes A, H, and R withappropriate concentrations are formulated, and then stored at −20° C. Inthe experiment, 200 μL of storage solution of enzyme H, 100 μL ofstorage solution of enzyme R and 25 μL of storage solution of enzyme Awere added to 4.7 mL of DMEM medium and then formulated into 5 mL ofdissociation solution for tumor tissues.

Example 2 Design of Flow Staining

Specific designs of different staining channels on cell surface areshown in Table 1.

TABLE 1 Channels used for cell surface molecular markers Channel BlankIsotype 2 Panel 2 FITC — Isotype Tim3 PE — Isotype PD-1 PerCP — CD4 CD4PE-Cy7 — Isotype Lag-3 APC — CD3 CD3 APC-Cy7 — CD8 CD8 BV421 Live/DeadLive/Dead Live/Dead BV510 — CD45 CD45

Example 3 Effects of Different Digestive Enzymes on the Positive Rate ofCheckpoint Proteins PD-1, TIM3 and LAG-3

The expression of PD-1, Tim-3 and Lag-3 in cytoxic T cells (CD8+T) andhelper T cells (CD4+T) was induced by PHA treatment. Whether or notdifferent digestive enzymes will affect expression levels of the threeproteins was analyzed in these two groups of cells.

The tumor is not a single cell suspension, thus it cannot be used inflow cytometry directly. If mechanical dissociation is used rather thanenzymatic dissociation, single-cell yield is relatively low, thereforepositive rate of single cell molecular marker proteins obtained bymechanical dissociation may not be able to represent the real value ofthe whole tissue. Thus, in the present disclosure, peripheral bloodmononuclear cells were used in the detection of molecular markerproteins of cells. It is confirmed that some dissociation reagent has aneffect on the expression level of marker proteins on cell surface.

First, cryopreserved human peripheral blood mononuclear cells (PBMC)were revived and then treated with 10 μg/mL of PHA for 48 hours to allowthe cells to be activated, followed by counting. The cells werealiquoted into 21 tubes; the number of cells is 3×10⁵ cells per tube. 5mL of dissociation buffer was added into each tube, while 5 mL of DMEMmedium (available from Gibco, Cat. No. 11960-051) was added intonegative control tube. The tubes were put into a 37° C. water bath(available from Shanghai Yiyou Company, model THZ-82), and the cellswere digested for 15 minutes. The specific information of differenttreatment groups are as followed:

TABLE 2 Dissociation conditions in different treatment groups NameDissociation Reagent Condition PBMC Negative control 37° C., 15 minutesMiltenyi Human Tumor Dissociation Kit 37° C., 15 minutes (Kit)Three-enzyme mixture 37° C., 15 minutes 1 mg/mL Collagenase D 37° C., 15minutes 100 μg/mL Hyaluronidase 37° C., 15 minutes 0.05 mg/mL DNase I37° C., 15 minutes Two-enzyme mixture 37° C., 15 minutes

The dissociated cells were centrifuged with a centrifuge (available fromEppendorf, model 5810R) and the supernatant was removed. The pelletswere washed twice with a phosphate buffer PBS (available from HycloneCorporation, Cat. No. SH3002802B) and centrifuged to remove thesupernatant, and then incubated with formulated antibody mixture at 4°C. for 30 minutes in dark.

The cells were centrifuged at 4° C., 300×g to remove the supernatant.The cells were resuspended in 200 μL of staining buffer for flowcytometry (available from BD Co., Cat. No. Pharmingen-554657) andcentrifuged at 4° C., 300×g for 5 minutes, and repeated once.

The cells were re-suspended in 100 μL of cell fixation buffer (availablefrom BD, Cat. No. BD-554655) and incubated at 4° C. for 20-30 minutes indark.

The cells were re-suspended in 200 μL of staining solution (availablefrom BD Co., Cat. No. Pharmingen-554657), centrifuged at 4° C., 300×gfor 5 minutes and repeated once, and finally re-suspended in stainingbuffer for flow cytometry (available from BD Company, Cat. No.Pharmingen-554657), and the suspension was transferred to a flow tubewith a final volume of 500 μL and detected with a cytometer (BD FACSCanto II).

The expression levels of Tim-3 and Lag-3 proteins in T cells (CD4+) andcytoxic T cells (CD8+) were significantly decreased after treatment withMiltenyi Human Tumor Dissociation Kit for 15 minutes as compared withthe negative control. Similarly, the expression levels of Lag-3 proteinin helper T cells (CD4+) and cytoxic T cells (CD8+) were significantlydecreased after treatment with three-enzyme mixtures or collagenase (asshown in FIG. 1, FIG. 2, FIG. 3 and TABLE 3).

In contrast, the expression of different cell surface molecular markerssuch as PD-1, Tim-3, and Lag-3 in the hyaluronidase or DNase I treatmentgroup was unchanged or not significantly decreased as compared with thenegative control group (as shown in FIG. 1, FIG. 2, FIG. 3 and TABLE 3).This suggests that the treatment of collagenase or Miltenyi Human TumorDissociation Kit will affect the expression levels of one or moremolecular markers on cell surface such as Tim-3 and Lag-3, which isdisadvantageous for further detection in flow cytometry. But thereagents not containing collagenase, such as hyaluronidase or DNase Ialone did not affect the expression of these immune cell surfacemolecular markers under given conditions. This experiment demonstratedthat neither DNase I at given dose nor hyaluronidase affected theexpression level of the checkpoint proteins. Furthermore, we found thatthe results of 1 mg/mL hyaluronidase or 1 μg/mL hyaluronidase treatmentwere consistent with that of 100 μg/mL hyaluronidase treatment thatneither one affected the level of cell surface molecular markers such asCD8, PD-1, Tim-3 and Lag-3. Similarly, the expression of PD-1, Tim-3 andLag-3 were not reduced after treatment with up to 50 μg/mL of DNase I.Although the fluorescence signal of CD8 was significantly reduced andPD-1 positive rate was slightly increased, the fluorescence signalintensities of other immunological checkpoints were not decreased. Andtreatment with less than 50 μg/mL of DNase I did not affect thedetection of molecular markers.

To further verify the protective effect of the hyaluronidase group orDNase I on the cell surface molecular markers, a mixture of DNase I andhyaluronidase was used to treat human peripheral blood mononuclear cells(PBMC) according to above experimental method. The experimental resultsindicated that the mixture of DNase I and hyaluronidase did not affectthe expression level of the checkpoint proteins (as shown in FIG. 4 andTable 4). This data was also advantageous in the increased selectivityfor enzyme species. It is anticipated that in certain tumor samples, thecell yield can be increased while the protein expression level isaltered.

TABLE 3 Positive expression rates (%) of different cell surfacemolecular markers PD- PD- Tim- Tim- Lag- Lag- Group CD4+ CD8+ 1+/CD4+1+/CD8+ 3+/CD4+ 3+/CD8+ 3+/CD4+ 3+/CD8+ Negative 55.80 22.80 53.30 58.4030.80 38.20 29.90 65.00 control Kit 49.10 27.10 59.90 62.60 7.99 11.702.74 13.50 Three- 53.70 25.80 58.30 60.50 31.40 35.40 6.05 22.10 enzymemixture Collage- 53.50 25.20 59.30 60.60 29.70 31.40 5.98 19.80 nase DHyaluron- 56.10 23.60 45.70 51.80 30.20 37.80 28.70 55.90 idase DNase I54.10 25.90 48.90 53.10 31.00 34.80 27.40 57.10

TABLE 4 Positive expression rate (%) of different cell surface molecularmarkers after treatment with the two-enzyme mixture PD- PD- Tim- Tim-Lag- Lag- Group CD4+ CD8+ 1+/CD4+ 1+/CD8+ 3+/CD4+ 3+/CD8+ 3+/CD4+3+/CD8+ Negative 33.7 25.9 59 49.4 40.6 55.2 53.2 79.9 control Two- 42.224 68.7 52.2 39.9 64.5 46.1 76.5 enzyme mixture

Example 4 Detection of Surface Marker CD8 on Human Peripheral BloodMononuclear Cells (PBMC)

Cryopreserved human peripheral blood mononuclear cells (PBMC) wererevived and then treated with 10 μg/mL of PHA for 48 hours to allow thecells to be activated, followed by counting. The cells were aliquoted,the number of cells is 3×10⁵ cells per tube. 5 mL of dissociation bufferwas added into each tube, while 5 mL of DMEM medium (available fromGibco, Cat. No. 11960-051) was added into negative control tube. Thetubes were put into a 37° C. water bath (available from Shanghai YiyouCompany, model THZ-82), and the cells were digested for 60 minutes.

The other steps were the same as that in Example 3, and it was foundthat the fluorescence intensity of CD8 was decreased after the treatmentwith the commercial human tumor kit (Accumax Cell Dissociation Solution)for 60 minutes, indicating that the kit treatment reduced the expressionlevel of CD8 (as shown in FIG. 5 and Table 5).

TABLE 5 Positive expression rates (%) of cell surface molecular markersCD4 and CD8 after treatment with Kit Group CD4+ CD8+ Negative control55.80 22.80 Kit; 60 minutes 53.70 13.90

Example 5 Treatment of Human Tumor Tissues

Clinical samples obtained by operation were placed in prepared MACStissue preservation solution and transported to WuXi AppTec Co. Ltd.(Shanghai). at 4° C. These tumor samples were treated within 48 hoursafter surgery. Before treatment of clinical tumor tissues, numbering ofthe corresponding sample was carried out, and the medical history of thepatient, texture and color of the tumor tissues, and clinicalinformation were recorded. Tumor tissues were weighed.

First, the obvious adipose tissue, fibrous tissue and necrotic part wereremoved, and then the resulting clinical tumor samples were washed threetimes in pre-cooled DMEM medium, finally the tumor tissue were cut into10 mm³ small pieces with ophthalmic scissors and tweezers.

50 μL of a solution of hyaluronidase with an initial concentration of 10mg/mL was added to 4.95 mL of DMEM medium (the final concentration is100 μs/mL) to prepare the dissociation reagent for tumor tissue, andthen 5 mL of prepared dissociation reagent for tumor tissue was put intoa C tube dedicated for gentleMACS Dissociator (available from MiltenyiCompany, Cat. No. 130-093-237), and the cut tumor tissues were alsotransferred to the C tube dedicated for Miltenyi tissue treatment withtweezers. After tightening of the lid, the tissue fragments in thedissociation reagent were gently shaken. Tumor tissues within the rangeof 10 mg to 1000 mg can be treated with this dissociation system.

The C tube was gently inserted into the C-tube slot of the gentleMACSDissociator (available from Miltenyi Company, Cat. No. 130-093-235). Andit should be noted that the tumor tissue fragments should beconcentrated at the blade area within the C tube.

The program was set to the h_tumor_01, and then run once. The C tube wasremoved after the end of the program h_tumor_01 and placed upward for awhile so as to place all tumor fragments in the dissociation solution atthe bottom of the tube. If necessary, the lid can be removed and thetissue adhered on the lid can be transferred with tweezers to the bottomdissociation reagent. The removed C tube was put into a 37° C. constanttemperature water bath for 7 minutes, which can be shook appropriatelyfor several times during the period. Repeat the above steps once.

The above C tube was gently inserted into the C-tube slot of theMiltenyi Tissue processor. The program was set to h_tumor_02, and thenrun twice. The C tube was removed and the tissue dissociation wasre-suspended in 20 mL of phosphate buffer. A 70 μm cell strainer(available from falcon, Cat. No. 352350) was placed on a 50 mLcentrifuge tube and the dissociated tissue was re-suspended and slowlypassed through the 70 μm cell strainer and, if necessary, the mincedfine tissue pieces can be grounded on the strainer to obtain moresingle-cell suspensions. The cell strainer was washed with 20 mL to 30mL of phosphate buffer so that the final volume of the single-cellsuspension obtained through the strainers was 50 mL.

The cells were centrifuged at 300×g for 10 minutes, and the supernatantwas removed with a pipette.

The single cells obtained in the previous step were re-suspended in 40mL of phosphate buffer and were centrifuged at 300×g for 7 min.

The cells were re-suspended into a single-cell suspension with 0.5 to 5mL of flow cytometry staining buffer, and counted by staining of trypanblue.

Example 6 Comparison of Cell Digestion Rates in Different Enzyme-TreatedGroups

The clinically obtained patient tumor samples were dissociated withdifferent digestive enzymes under the same temperature and timeconditions according to the method of Example 5, and the single-cellyield was counted with trypan blue staining. Each sample was countedthree times, and the average number+SEM of single cells per gram oftumor was shown in the figure.

In FIG. 6, although the cell yield varies depending on the tumor speciesor states between different samples, it is comparable between thecombination of the three enzymes involved in the present disclosure andthe conventional enzyme combination methods reported in the literature,and the differences of cell yield were not large, thus the resultingcells were sufficient for further flow cytometry analysis.

Although the present disclosure is not limited thereto, it will beunderstood by those skilled in the art that various modifications andvariations can be made within the scope of the present disclosure, themanner of changes are also within the scope of the present disclosure.

1. A tumor dissociation reagent which does not comprise collagenase butcomprises hyaluronidase, wherein the tumor dissociation reagent does notdegrade or partially degrade membrane surface receptor.
 2. The tumordissociation reagent of claim 1, which further does not comprisetrypsin.
 3. The tumor dissociation reagent of claim 1, which furthercomprises DNase I.
 4. The tumor dissociation reagent of claim 1, whereinthe membrane surface receptor is a checkpoint receptor.
 5. The tumordissociation reagent of claim 1, wherein the membrane surface receptorcomprises CD8, PD-1, PD-L1, TIM-3 or LAG-3 protein.
 6. A method fordetecting protein expression level of an immunological checkpoint markerin tumor tissue, comprising applying the tumor dissociation reagent ofclaim 1 to the tumor tissue.
 7. The method of claim 6, wherein the tumordissociation reagent further comprises DNase I.
 8. The method of claim6, wherein the tumor tissue includes tumor infiltrating immune cell. 9.The method of claim 6, wherein the protein expression in tumor tissue isdetected by flow cytometry.
 10. The method of claim 6, wherein theprotein is membrane surface receptor.
 11. The method of claim 10,wherein the membrane surface receptor comprises CD8, PD-1, PD-L1, TIM-3or LAG-3 protein.
 12. A kit for tumor dissociation comprising the tumordissociation reagent of claim
 1. 13. The kit of claim 12, wherein thetumor dissociation reagent further comprises DNase I.
 14. A method ofdetecting protein expression in tumor tissue by flow cytometrycomprising applying the kit of claim 12 to the tumor tissue.
 15. Amethod of preventing degradation of an immunological checkpoint markerin tumor tissue, comprising treating cells with the tumor dissociationagent of claim
 1. 16. The method of claim 15, wherein the tumordissociation reagent further comprises DNase I.
 17. The method of claim15, comprising dissolving the tumor tissue with the tumor dissociationreagent, and detecting the immunological checkpoint marker by flowcytometry.
 18. The method of claim 17, wherein the tumor tissue includestumor infiltrating immune cell.